LncRNA LINC01232 Enhances Proliferation, Angiogenesis, Migration and Invasion of Colon Adenocarcinoma Cells by Downregulating miR-181a-5p

LncRNAs play crucial roles in the progression of colon adenocarcinoma (COAD), but the role of LINC01232 in COAD has not received much attention. The present study was designed to explore the related mechanisms of LINC01232 in the progression of COAD. LINC01232, miR-181a-5p, p53, c-myc, Bcl-2, cyclin D1, p16, Bax, VEGF, E-cadherin, vimentin, N-cadherin and SDAD1 expressions were determined by western blot and qRT-PCR. CCK-8, tubule formation, and Transwell assays were employed to detect proliferation, angiogenesis, and migration/invasion of COAD cells, respectively. The relationship between LINC01232 and miR-181a-5p was predicted by LncBase Predicted v.2, and then verified through dual luciferase reporter gene assay. According to the results, LINC01232 was highly expressed in COAD cells and enhanced proliferation, angiogenesis, migration, and invasion of COAD cells. Downregulated LINC01232 promoted expression of p53 and p16, and inhibited c-myc, Bcl-2 and cyclin D1 expressions in COAD cells, while upregulation of LINC01232 generated the opposite effects. LINC01232 was negatively correlated with miR-181a-5p while downregulated miR-181a-5p could reverse the effects of siLINC01232 on cell proliferation, angiogenesis, migration, and invasion. Similarly, miR-181a-5p mimic could also offset the effect of LINC01232 overexpression. SiLINC01232 increased the expressions of Bax and E-cadherin, and decreased the expressions of VEGF, vimentin, N-cadherin and SDAD1, which were partially attenuated by miR-181a-5p inhibitor. Collectively, LINC01232 enhances the proliferation, migration, invasion, and angiogenesis of COAD cells by decreasing miR-181a-5p expression.

pancreatic cancer by regulating TM9SF2 in pancreatic adenocarcinoma progression [12]. Similarly, Meng et al. demonstrated that LINC01232 is a promising target molecule for pancreatic cancer treatment [13]. However, the role of LINC01232 in COAD is unclear.
One way in which lncRNAs participate in cellular biological processes is through competitive binding to target micro (mi)RNAs, thereby affecting the expressions of downstream genes [14,15]. MiRNAs are also a kind of noncoding RNA, and have aroused considerable concern among researchers in recent years. According to a previous study, LINC01232 could competitively bind to miR-654-3p, and reduce its expression in ESCC cells, thus promoting the expression of HDGF [16]. Nevertheless, the regulation of miRNA by LINC01232 in colon cancer is still undefined. Through bioinformatics analysis, we found that LINC01232 could bind to miR-181a-5p. It is noteworthy that the involvement of miR-181a-5p in the progression of COAD has been confirmed [17,18]. On this basis, we hypothesized that LINC01232 affects the progression of COAD by modulating miR-181a-5p.
In the present study, we first measured the expressions of LINC01232 and miR-181a-5p in COAD and normal cells. The roles of LINC01232 and miR-181a-5p in the progression of COAD were then determined by regulating their expressions. Therefore, our study may provide a significant therapeutic target for the treatment of COAD.

qRT-PCR
Total RNA of cells was extracted by Trizol reagent (15596-018, Invitrogen), the concentration of which was determined by a microplate reader (Molecular Devices, USA). Next, 1 μg RNA was reversely transcribed into Table 1. Specific primer sequences for quantitative reverse transcription polymerase chain reaction.

Gene
Primer sequence Species miR-181a-5p Human cDNA, which was used for qPCR, with a First cDNA Synthesis Kit (RR037A, TaKaRa, Japan). In short, 20 μl reaction solution was prepared as follows: 2 μl cDNA, 10 μl SYBR Mix (RR820A, TaKaRa), 0.8 μl forward primer, 0.8 μl reverse primer and 6.4 μl sterile water. Then, the mixture was amplified under the following reaction conditions: 95°C for 30 s, 40 cycles of 95°C for 3 s, and 60°C for 30 s. The primers were obtained from Sangon (China) and RIBIBIO, and the sequences were listed in Table 1. U6 and β-actin were internal references. Finally, the CT value obtained from a 7900 Real-Time PCR System (Biosystems, USA) was calculated by 2 -ΔΔCT method [19].

Cell Counting Kit (CCK)-8
After transfection for 24 h, SW-620 and LOVO cells were inoculated in a 96-well plate (3000/well). Cell culture was continued for another 24 and 48 h, and the CCK-8 assay was carried out. Then, 10 μl CCK-8 reagent (C0038, Beyotime, China) was added to each well, mixed with cells gently, and placed in the incubator for 3 h. At the end of incubation, the culture plate was taken out and placed in a microplate reader to detect the light absorption at 450 nm.

Tubule Formation Assay
Fifty microliters of matrix glue (356234, Becton, Dickinson and Company, USA) was added to a 6-well plate, which was then incubated for 30 min. After transfection for 24 h, SW-620 and LOVO cells were collected and resuspended in 2 ml medium. Then, 50 μl cells were added to the pre-coagulated matrix glue for further culture in the incubator. After 12 h, the culture plate was photographed under an inverted microscope (POMEAS, China), and Image J (1.8.0, National Institutes of Health, Germany) was used to calculate the length of the tubule.

Transwell Assay
Transfected or untransfected cells were resuspended in 2 ml medium without FBS. For invasion detection, the upper chamber of the Transwell plate (3428, Corning, USA) was first uniformly coated with the matrigel (Corning) and placed in the incubator for 4 h. Then, 100 μl pre-prepared cells were put into the upper chamber, and 750 μl medium containing 10% FBS was put into the lower chamber. Following culture for 48 h, the cells of the upper chamber were gently removed with a cotton swab. Cells invading the lower chamber were immersed in 4% paraformaldehyde (P0099, Beyotime) for 30 min, and then stained by crystal violet (C0121, Beyotime) for 30 min. Next, the invading cells were photographed with an inverted microscope, and 5 fields were randomly selected to count the number of cells. The Transwell assay for migration detection was the same as above except that matrigel was not required.

Statistical Analysis
Data were expressed as the means ± SD. Differences among groups were determined statistically using analysis of variance (ANOVA). Statistical analyses were performed by the SPSS software (19.0, IBM, USA). p < 0.05 indicated statistically significant difference.

LINC01232 Was Highly Expressed in COAD Cells
For the first time, we identified the expression of LINC01232 in COAD cells. According to Fig. 1A, the qRT-PCR detection results showed the expression of LINC01232 in COAD cells to be higher than that in normal colon fibroblasts (p < 0.05, p < 0.01, p < 0.001). As the expression of LINC01232 was the highest in SW-620 cells and the lowest in LOVO cells, the two cells were selected as subjects for further experiments. In addition, due to the high expression level of LINC01232 in SW-620 cells, we transfected siLINC01232 into SW-620 cells to downregulate the level of LINC01232, and thus unveil its role (Fig. 1B, p < 0.001). By contrast, we then upregulated LINC01232 expression in LOVO cells through transfection of LINC01232 overexpression plasmid, and the transfection efficiency was exhibited in Fig. 1C (p < 0.001).

LINC01232 Was Negatively Correlated with miR-181a-5p
LncBase Predicted v.2 was employed to predict the binding site of LINC01232 and miR-181a-5p, and the LINC01232 sequence of the binding site mutation was designed (Fig. 4A). Notably, the prediction result was verified by dual-luciferase reporter gene assay. Co-transfection of LINC01232-WT and miR-181a-5p mimic could reduce the luciferase activity of cells, while co-transfection of LINC01232-MUT and miR-181a-5p mimic had no significant effect on the luciferase activity of cells, as compared with their control (Figs. 4B and 4C).  Subsequently, we found that downregulation of LINC01232 enhanced, but upregulation of LINC01232 inhibited the expression of miR-181a-5p, when compared with their control (Figs. 4D and4E, p < 0.001).

Discussion
Colon adenocarcinoma is one of the most common cancers of the digestive tract globally, and lncRNAs have been evidenced to play vital roles in colon carcinogenesis and progression [20]. In this study, we found that the expression of LINC01232 was different in various cell lines, and abnormally high in COAD cells. We believed that this difference may stem from the different origins of cell lines, and the different phenotypes of each cell, which enable them to create the appropriate microenvironment [21]. Metastasis and invasion are the leading causes of death in COAD patients [22]. Here, we explored the relationship between LINC01232 and COAD cell migration and invasion by silencing or overexpressing LINC01232. From the results of Transwell assay, we could conclude that LINC01232 silencing hindered cell migration and invasion, and that LINC01232 overexpression had opposite effects. This suggested that LINC01232 was indeed involved in the migration and invasion of COAD p53, Bcl-2, Bax, VEGF, vimentin, E-cadherin, N-cadherin and SDAD1 in SW-620 and LOVO cells transfected or untransfected with siLINC01232, LINC01232 overexpression plasmid, miR-181a-5p mimic and miR-181a-5p inhibitor were determined by qRT-PCR or western blot. β-Actin served as an internal reference. * vs. IC+siNC, ^v s. I+siNC, # vs. IC+siLINC01232, & vs. MC+NC, Δ vs. M+NC, † vs. MC+LINC01232; * or # or & or † p < 0.05, ** or^^ or ## or && or ΔΔ or † † p < 0.01, *** or^^^ or ### or &&& or ΔΔΔ or † † † p < 0.001. p53, protein 53; Bcl-2, B-cell lymphoma-2; VEGF, vascular endothelial growth factor; SDAD1, SDA1 domain containing 1; Bax, Bcl-2-associated X; I, miR-181a-5p inhibitor; IC, inhibitor control; M, miR-181a-5p mimic; MC, mimic control; siNC, siRNA negative control.
cells. In addition, we also unraveled that LINC01232 overexpression enhanced the proliferation of COAD cells. Notably, abnormal cell proliferation is the basis of tumorigenesis [23]. Thus, LINC01232 has been confirmed to be implicated in the progression of colon cancer in our study.
To further verify the results of the above experiments, the expressions of p53, p16, c-myc, Bcl-2, and cyclin D1 were quantitated. P53 is a tumor suppressor gene that is mutated in about 50% of malignant tumors [24]. Thus, p53 could be used as a predictor of the progression from precancerous lesions to true malignant tumors [25]. Besides, an earlier study also proved that the expression of p53 was closely related to the invasion and lymphatic metastasis of COAD [26]. Likewise, p16 gene is also a critical tumor suppressor gene, which will lead to the proliferation of malignant cells after inactivation [27]. In line with a recent study, abnormality in the cyclinD1-CDK-p16-pRb pathway is the genetic basis of tumor development [28]. Overexpression of cyclin D1 protein will enhance the binding of cyclin D1 to cyclin-dependent kinases, further stimulate cell division, promote excessive cell proliferation, inhibit cell apoptosis, and finally lead to carcinogenesis [29]. Furthermore, as a nuclear transcription factor, c-myc could promote cell proliferation, enable cells at rest to enter into proliferation, and transform cells into undergoing malignant changes [30]. Predictably, we examined the expression of Bcl-2, a wellknown apoptotic inhibitor [31]. In light of the data, overexpression of LINC01232 could decrease p53 and p16 expressions, and increase c-myc, Bcl-2 and cyclin D1 expressions. These results further confirmed that LINC01232 is involved in the proliferation, migration, and invasion of COAD cells. Furthermore, we noted that LINC01232 affected angiogenesis in colon cancer cells. It is worth mentioning that angiogenesis is vital for the rapid growth and metastasis of solid tumors [32]. Our results suggested that LINC01232 silencing attenuated, but LINC01232 overexpression strengthened, the tubule formation of COAD cells. The angiogenesis promoted by LINC01232 creates a strong condition for the development of COAD.
MiR-181a-5p, a targeted binding molecule downstream gene of LINC01232, has been demonstrated to be associated with the progression of COAD [17,18]. The targeting relationship between miR-181a-5p and LINC01232 has also been verified in our study. We also corroborated that miR-181a-5p was dramatically lowly expressed in COAD [18,33]. To further explore the roles of miR-181a-5p and LINC01232 in the progression of COAD, we simultaneously knocked down or overexpressed miR-181a-5p and LINC01232 in COAD cells. As previously mentioned, LINC01232 knockdown would dampen COAD cell proliferation, migration, invasion, and angiogenesis. Expectedly, downregulation of miR-181a-5p boosted proliferation, migration, invasion, and angiogenesis of COAD cells. In addition, downregulation/upregulation of miR-181a-5p could attenuate the effects of LINC01232 silencing/overexpression on the proliferation, migration, invasion, and angiogenesis of COAD cells. This implied that LINC01232 could enhance the proliferation, migration, invasion, and angiogenesis of COAD cells by inhibiting miR-181a-5p expression.
Then, we continued to verify the above results by detecting proliferation-, migration-, invasion-, and angiogenesis-related proteins. In addition to p53 and Bcl-2 expressions, Bax, VEGF, E-cadherin, vimentin, Ncadherin and SDAD1 expressions were also detected. Bax is a well-known pro-apoptotic protein in contrast to Bcl-2, an anti-apoptotic protein [34]. Particularly, VEGF is a functional glycoprotein with high biological activity [35]. It is the only growth factor specifically acting on vascular endothelial cells, and is most directly involved in inducing tumor angiogenesis and enhancing vascular permeability [33]. Besides, E-cadherin, vimentin, and Ncadherin are all epithelial-mesenchymal transformation (EMT)-related proteins. EMT means that under certain conditions, epithelial cells lose their epithelial phenotypic characteristics and connections to each other, acquire stromal-like characteristics and motor ability, and can leave the in situ tissue [36]. EMT can enable stationary tumor cells to acquire motor ability, making tumor metastasis possible [37]. Furthermore, SDAD1 promotes the proliferation of COAD cells by reducing apoptosis [38]. Our results corroborated that the downregulated LINC01232 could promote p53, Bax and E-cadherin expressions, while suppressing Bcl-2, VEGF, vimentin, Ncadherin, and SDAD1 expressions, but such effects were reversed by miR-181a-5p inhibitor.
In summary, this study provides an overview of the role of LINC01232 in regulating COAD cell proliferation, migration, invasion, and angiogenesis. Further discussion revealed that the influence of LINC01232 on COAD progression is achieved by downregulating miR-181a-5p level. Our results may provide a potential therapeutic target for the treatment of COAD.